Use of lactobacillus salivarius

ABSTRACT

Lactobacillus salivarius  is useful in the prophylaxis or treatment of undesirable inflammatory activity, especially gastrointestinal inflammatory activity such as inflammatory bowel disease or irritable bowel syndrome. The inflammatory activity may also be due to cancer. The  Lactobacillus salivarius  is of human origin isolated from resected and washed human gastrointestinal tract. One such strain is UCC 118 described in WO-A-9835014.

This is a Continuation of application Ser. No. 10/804,223, filed Mar.19, 2004, which in turn is a Continuation of application Ser. No.10/241,797, filed Sept. 12, 2002, which in turn is a Continuation ofapplication Ser. No. 09/903,590, filed Jul. 13, 2001, now abandoned;which in turn is a Continuation of PCT/IE00/00007, filed Jan. 17, 2000.

INTRODUCTION

The invention relates to the use of strains of Lactobacillus salivarius.

The defense mechanisms to protect the human gastrointestinal tract fromcolonization by intestinal bacteria are highly complex and involve bothimmunological and non-immunological aspects (V. J. McCracken and H. RGaskins, ‘Probiotics a critical review’, Horizon Scientific Press, UK,1999, p 278.). Innate deference mechanisms include the low pH of thestomach, bile salts, peristalsis, much layers and anti-microbialcompounds such as lysozyme (D. C. Savage, ‘Microbial Ecology of theGut’, Academic Press, London, 1997, p.278.). Immunological mechanismsinclude specialised lymphoid aggregates, underlying M cells, calledpeyers patches which are distributed throughout the small intestine andcolon (M. F. Kagnoff. Gastrocaterol. 1993, 105, 1275). Luminal antigenspresented at these sites result in stimulation of appropriate T and Bcell subsets with establishment of cytoline networks and secretion ofantibodies into the gastrointestinal tract (M. R. Neutra and J-PKraehenbuhl, ‘Essentials of mucosal immunology’, Academic Press, SanDiego, 1996, p.29., M. E. Lamm. Ann. Rev. Mirobiol. 1997, 51, 311). Inaddition, antigen presentation may occur via epithelial cells tointraepithelial lymphocytes and to the underlying lamina propria immunecells (S. Raychaudhur et al. Nat Biotecinol., 1998, 16, 1025).Therefore, the host invests substantially in immunological defense ofthe gastrointestinal tract. However, as the gastrointestinal raucous isthe largest surface at which the host interacts with the externalenvironment, specific control mechanisms must be in place to regulateimmune responsiveness to the 100 tons of food which is handled by thegastrointestinal tract over an average lifetime (F. Shanahan,‘Physiology of the gastrointestinal tract’, Raven Press, 1994, p.643.).Furthermore, the gut is colonised by over 500 species of bacterianumbering 10¹¹-10 ¹²/g in the colon. Thus, these control mechanisms mustbe capable of distinguishing non-pathogenic adherent bacteria frominvasive pathogens which would cause significant damage to the host. Infact, the intestinal flora contributes to defence of the host bycompeting with newly ingested potentially pathogenic micro-organisms.

Consumption of non-pathogenic, or probiotic, bacteria has resulted inenhancement of immune parameters in healthy volunteers. Examples ofthese immune modulatory effects are given in Table 1. TABLE 1 ImmuneEnhancing Effects Following Oral Consumption of Probiotic Bacteria.Observed Effect Reference Increased Macrophage Phagocytosis 10 IncreasedNatural Killer Cell Activity 11 Increased IFNγ serum levels 12 IncreasedB cell and NK cell numbers 12 Promotion of IgA responses 11, 13-15Increased DTH responses 16

Bacteria present in the human gastrointestinal tract can promoteinflammation. Aberrant immune responses to the indigenous microflorahave been implicated in certain disease states, such as inflammatorybowel disease (Brandzeag P. et al. Springer Semin. Immunopathol., 1997,18, 555). Antigens associated with the normal flora usually lead toimmunological tolerance and failure to achieve this tolerance is a majormechanism of mucosal inflammation (Stallmach A. et al., Immunol. Today,1998, 19, 438). Evidence for this breakdown in tolerance includes anincrease in antibody levels directed against the gut flora in patientswith lBD. WO-A-98/35014 describes strains of Lactobacillus salivariusisolated from resected and washed human gastrointestinal tract whichinhibits a broad range of Gram positive and Gram negative microorganismsand which secretes a product having anti-microbial activity into acell-free supernatant.

STATEMENTS OF INVENTION

The immune system is designed to defend host tissue and destroy invadingpathogens. Upon recognition of the presence of a bacterial cell, cellsof the immune system become activated and eliminate the bacterialthreat. The production of inflammatory mediators promote cellularactivation and pathogen destruction.

Surprisingly, we have found that strains of Lactobacillus salivariuselicit an anti-inflammatory effect in viro and in vivo. We have foundthat the immune perception of Lactobacillus salivarius results in thesuppression of inflammatory activity. The deliberate consumption inlarge numbers of Lactobacillus salivarius results in the suppression ofinflammatory activity. The invention is therefore of major potentialtherapeutic value in the prophylaxis or treatment of undesirableinflammatory responses, such as inflammatory bowel disease.

Lactobacillus salivarius is a commensal microorganism originallyisolated from the microbial flora within the human gastrointestinaltract. The immune system within the gastrointestinal tract cannot have apronounced reaction to members of this flora as the resultinginflammatory activity would also destroy host cells and tissue function.Therefore, some mechanism(s) exist whereby the immune system canrecognise commensal non-pathogenic members of the gastrointestinal floraas being different to pathogenic organisms. This ensures that damage tohost tissues is restricted and a defensive barrier is still maintained.

According to the invention there is provided use of a strain ofLactobacillus salivarius in the prophylaxis and/or treatment ofundesirable inflammatory activity.

The invention the undesirable inflammatory activity may be undesirablegastrointestinal inflammatory activity such as inflammatory boweldisease, eg. Crohns disease, ulcerative colitis, irritable bowelsyndrome, pouchitis or post infection colitis.

The gastrointestinal inflammatory activity may also be diarrhoealdisease. The diarrhoeal disease may be associated by Clostridiumdifficile or be associated with Rotovirus. The diarrhoeal disease mayalso be post infective diarrhoeal disease.

The inflammatory activity may be due to gastrointestinal cancer orsystemic inflammatory disease such as rheumatoid arthritis.

In another instance the undesirable inflammatory activity may be due toan autoimmune disorder.

In yet another instance the undesirable inflammatory activity may be dueto cancer.

In one embodiment the invention provides use of a strain ofLactobacillus salivarius in the prophylaxis of cancer.

In another embodiment the invention provides use of a strain ofLactobacillus salivarius wherein the Lactobacillus salivarius iscontained in a formulation.

Preferably the formulation includes another probiotic material.Alternatively or additionally the formulation includes a prebioticmaterial.

Ideally the formulation includes an ingestable carrier. The ingestablecarrier may be a pharmaceutically acceptable carrier such as a tablet,capsule or powder.

Preferably the ingestable carrier is a protein and/or a peptide, inparticular proteins and/or peptides that are rich inglutamine/glutamate; a lipid; a carbohydrate; a vitamin; mineral and/ortrace element.

Most preferably the ingestable carrier is a food product such asacidified milk, yoghurt, frozen yoghurt, milk powder, milk concentrate,cheese spreads, dressings or beverages.

In one embodiment the Lactobacillus salivarius is present in theformulation at more than 10° cfu per gram of delivery system.

In another embodiment the formulation includes an adjuvant. Theformulation may include a bacterial component. The formulation mayalternatively or additionally include a drug entity. The formulation mayalso include a biological compound.

In one embodiment the invention provides use of a strain ofLactobacillus salivarius wherein the strain or formulation is foradministration to animals. Preferably the animal is a mammal, mostpreferably a human.

In another embodiment the invention provides use of a strain ofLactobacillus salivarius wherein the Lactobacillus salivarius effectschanges in an immunological marker when introduced into a systemcomprising cells which interact with the immune system and cells of theimmune system.

Preferably the cells which interact with the immune system areepithelial cells. Most preferably the immunological marker is a cytokineespecially TNFα.

Preferably the cells which interact with the immune system and theimmune system cells are of matched origin.

In one embodiment the cells which interact with the immune system are ofgastrointestinal, respiratory or genitourinary origin.

In another embodiment the cells of the immune system are ofgastrointestinal, respiratory or genitourinary origin. In a furtherembodiment the invention provides use of a strain of Lactobacillussalivarius wherein the Lactobacillus salivarius strain is Lactobacillussalivarius subspecies salivarius. Preferably the Lactobacillussalivarius is of human origin, most preferably from resected and washedhuman gastrointestinal tract.

Preferably the Lactobacillus salivarius inhibits a broad range of Grampositive and Gram negative micro-organisms. Most preferably it secretesa product having anti-microbial activity into a cell-free supernatant,said activity being produced only by growing cells and being destroyedby proteinase K and pronase E.

An especially preferred strain of Lactobacillus salivarius isLactobacillus salivarius strain UCC 188 or mutant or variant thereof.

A deposit of Lactobacillus salivarius strain UCC 118 was made at theNCIMB on Nov. 27, 1996 and accorded the accession number NCIMB 40829.The strain of Lactobacillus salivarius is described in WO-A-98/35014.

The Lactobacillus salivarius may be a genetically modified mutant or itmay be a naturally occurring variant of Lactobacillus salivarius.

Preferably the Lactobacillus salivarius is in the form of viable cells.Alternatively the Lactobacillus salivarius may be in the form ofnon-viable cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of C. perfringens levels in the mice consuming UCC118compared to a placebo group (p<0.05). Results are plotted as the meanlog values±standard error for each of the groups

FIG. 2 is a bar chart of inflammatory scores for mice consuming UCC118in comparison to control mice. Results are shown as the mean±standarderror for each of the groups.

FIG. 3 is a graph of TNFα levels over six weeks that patients consumeUCC118. Results are plotted as the mean pg/ml TNFα level for each timepoint (n=22).

FIG. 4 is a graph of CDAI scores for patients consuming UCC118 over thecourse of probiotic feeding. CDAI scores decreased from an average of180 to 160.

FIG. 5 is a graph of cytokine production in vitro following exposure toUCC118, Results are expressed as pg/ml.

FIG. 6 is bar chart of extracellular TNFα, IL-1RA, IL-6, sIL-6R, andIFNα levels following exposure to Lactobacillus salivarius UCC118.

FIG. 7 is a gene array with specific gene sequences for 268 cytokinesand related molecules to examine the immune response to UCC118. Thebottom panel illustrates the control culture while the top panelillustrates cytokine gene expression by PBMCs following exposure-to UCC118; and

FIG. 8 is a bar chart of TNFα levels in the presence of variousbacterial strains.

DETAILED DESCRIPTION

We have developed criteria for in vitro selection of probiotic bacteriathat reflect certain in vivo effects on their host, such as modulationof the GIT microflora and modulation of the mucosal immune responseresulting in the production of secretory antibodies specific to theconsumed strain. We have found that Lactobacillus salivarius subsp.salivarius UCC118 not only survives passage through the gastrointestinaltract and adheres to human intestinal cell lines but also, surprisinglyhas anti-inflammatory effects.

The general use of probiotic bacteria is in the form of viable cells.However, it can also be extended to non-viable cells such as killedcultures or compositions containing beneficial factors expressed by theprobiotic bacteria. This could include thermally killed micro-organismsor micro-organisms killed by exposure to altered pH or subjection topressure. With non-viable cells product preparation is simpler, cellsmay be incorporated easily into pharmaceuticals and storage requirementsare much less limited than viable cells. Lactobacillus caseiYIT 9018offers an example of the effective use of heat killed cells as a methodfor the treatment and/or prevention of tumour growth as described inU.S. Pat. No. 4,347,240.

It is unknown whether intact bacteria are required to exert ananti-inflammatory effect or if individual active components of theinvention can be utilised alone. Proinflammatory components of certainbacterial strains have been identified. The proinflammatory effects ofgram-negative bacteria are mediated by lipopolysaccharide (LPS). LPSalone induces a proinflammatory network, partially due to LPS binding tothe CD14 receptor on monocytes. It is assumed that components ofprobiotic bacteria possess anti-inflammatory activity, due to theeffects of the whole cell. Upon isolation of these components,pharmaceutical grade manipulation is anticipated.

The invention will be more clearly understood from the followingexamples.

EXAMPLE 1

Detailed description of the in vivo demonstration of theanti-inflammatory effects of Lactobacillus salivarius especiallysubspecies salivarius UCC118.

Murine Model of Gastrointestinal Inflammation

Aberrant immune responses to the indigenous microflora have beenimplicated in certain disease states, such as inflammatory bowel disease(Brandzeag P., et al. Springer Semin. Immunopathol., 1997, 18, 555).Antigens associated with the normal flora usually lead to immunologicaltolerance and failure to achieve this tolerance is a major mechanism ofmucosal inflammation (Stallmach A., et al. Immunol. Today, 1998, 19,438). Evidence for this breakdown in tolerance includes an increase inantibody levels directed against the gut flora in patients with IBD. Inaddition, certain mouse models predisposed to inflammatory lesions inthe gastrointestinal tract remain disease free when housed in germ freeconditions or when treated with antibiotics (Kuhn R., et al. Cell, 1993,75, 263; Panwala C. M., et al. J.Immunol., 1998, 161, 5733).

C57BL/6 Interleukin-10 knockout mice are predisposed to developingenterocolitis in the presence of an enteric bacterial flora. Whenmaintained in germ free conditions, IL-10 knock out mice do not developthe disease (Kuhn R., et al. Cell, 1993, 75, 263). Since thepathogenesis of this disease has been linked with the enteric flora,elimination of specific components of this flora may have a beneficialeffect on the severity of this disease.

Lactobacillus salivarius subsp. salivarius UCC118 is a probioticbacteria, which was isolated from a healthy human ileum. It is suited togastrointestinal colonization as it fulfills many criteria set down forthe selection of probiotic strains. These include traits such as biletolerance, acid resistance and in vitro adherence to human colonic celllines. Feeding trials in healthy humans have been conducted andconsiderable modification of the gastrointestinal flora was noted. Inaddition, UCC118 was perceived by the mucosal immune system resulting inthe production and secretion of IgA specific to UCC118.

Thus, UCC118 survives passage through the gastrointestinal tract,modulates the gut flora and is perceived by the mucosal immune system.The influence of this probiotic bacteria in modulating inflammatoryresponses within the gastrointestinal tract was examined using a murinemodel of enterocolitis. In addition, we examined the role ofLactobacillus salivarius subsp. salivarius UCC118 in reducing the rateof neoplastic change within the gastrointestinal tract.

Twenty IL-10 KO mice were studied (ten consumed probiotic organisms inmilk and 10 consumed unmodified milk) for 16 weeks. Fecal microbialanalysis was performed weekly to enumerate excretion of lactobacilli,Clostridium perfinges, bacteroides, coliforms, bifidobacteria andenterococci. At sacrifice, small and large bowel were microbiologicallyand histologically assessed.

Fecal coliform and enterococci levels were significantly reduced in testanimals compared to the controls. At sacrifice, a significant reductionin C. perfringens numbers was observed in the test mice (FIG. 1). Therewere no fatalities in the test group compared to two deaths fromfulminant colitis in the control group. Only one test mouse developedcolonic adenocarcinoma compared to five in the control group. Testanimal mucosal inflammation consistently scored lower than that of thecontrol mice (FIG. 2). The reduction in tumour incidence followingconsumption of UCC118 may be related to the reduced level ofinflammation within the gastrointestinal tract or may be due toelimination of pro-carcinogenic members of the gastrointestinal flora(Rumney C. J., et al. Carcinogenesis, 1993, 14, 79; Rowland I. R.(1995). In: Gibson G. R. (ed). Human colonic bacteria: role innutrition. physiology and pathology, pp 155-174. Boca Raton CRC Press;Darveau D. Nat. Biotech., 1999, 17, 19).

In conclusion, consumption of Lactobacillus salivarius UCC 118 resultsin a significant modulation of the gut flora and an improvement inmortality rate, cancer incidence and disease score.

EXAMPLE 2 Human Trial with UCC118 in Patients with Active Crohn'sDisease.

Inflammatory bowel disease (IBD) encompasses a number of inflammatorydisorders of the gastrointestinal tract, including both Crohn's diseaseand ulcerative colitis.

Patients suffering from active Crohn's disease have been treated withUCC118. Briefly, UCC118 was consumed in a fermented milk product for 6weeks by 22 patients. Microbiological and immunological determinationswere made at week 0, week 1, week 3 and week 6. This was not aplacebo-controlled trial.

A number of systemic cytoline levels were measured over the course offeeding. In particular, tumour necrosis factor α(TNFα), aproinflammatory cytokine that has been implicated in the pathogenesis ofmany inflammatory disease states, including inflammatory bowel disease.Current therapies for inflammatory bowel disease specifically aim toreduce TNFα levels (Present. D. H., et al. New Eng. J. Med., 1999, 340,1398). In this trial, systemic TNFα levels were reduced followingconsumption of UCC118 (FIG. 3).

In addition, patients were assessed regarding their Crohn's DiseaseActivity Index (CDAI) over the six week trial period. This indexassesses the general health and well being of each patient (FIG. 4).Overall, the disease activity index improved slightly for the majorityof individuals in the trial. These are patients with moderatelyactive-disease and their CDAI scores would be expected to increase.However, following treatment with UCC118, CDAI scores did not increaseand in fact they improved from a mean of 180 to 160.

EXAMPLE 3 Detailed Description of the in vitro Demonstration of theMechanisms Underlying the Anti-inflammatory Effects of Lactobacillussalivarius Especially subspecies salivarius UCC118.

A number of methodologies have been utilised for these studies includingELISAs (extracellular protein determination), flow cytometry(intracellular protein determination) and cDNA expression arrays (mRNAexpression). In particular, examination of the expression of tumournecrosis factor α has been targeted, due to its clinical importance, andsuppression of the production of this cytokine, following exposure toUCC118, has been noted using all three methodologies.

Using a transwell assay system, with epithelial cells and peripheralblood mononuclear cells, extracellular cytoline levels were measured byELISAs. Following co-incubation with UCC118, the amount of TNFα producedwas significantly reduced compared to control cultures. Furthermore,IL-1RA and IFN-γ levels dropped while IL6 and soluble IL-6 receptorlevels increased (FIG. 5). Intracellular staining for TNFα confirmed theELISA result as TNFα levels were lower-in the UCC118 stimulated samplecompared to controls.

FIG. 6 demonstrates the tricellular signalling that occurs.Co-incubation of PBMCs and Lactobacillus salivarius strain UCC118results in the stimulation of TNFα production. However, co-incubation ofPBMCs, Lactobacillus salivarius strain UCC118 and epithelial cells(CaCo-2 cells) results in a significant inhibition of TNFα production.Thus, a significantly different pattern of signalling is present in thetricellular model compared to bacteria and PBMCs alone.

Gene arrays measure the quantity of mRNA in a population of cells. Westimulated peripheral blood mononuclear cells with UCC118 for 24 hoursand we examined the effect on cytoline gene expression (FIG. 7).Considerable modification of cytokine gene expression was noted. Forexample, genes encoding the proinflammatory cytokines IL-1β and TNFαwere turned off while genes encoding Th2 type cytokines, such as IL-6,were enhanced.

in vitro models have demonstrated that UCC118 is capable of inducing Th2type cytokines (i.e. IL-6 and IL-6 soluble receptor) while suppressingthe production of inflammatory cytokines such as TNFα and IL-1β. Thus,these results suggest that consumption of UCC118 would be of benefit topatients suffering from inflammatory diseases, such as IBD.

EXAMPLE 4 Test for Anti-inflammatory Bacterial Strains

A number of lactic acid bacteria, which have been isolated from thehuman gastrointestinal tract, were examined in this novel assay systemfor anti-inflammatory effect. All bacterial strains were taken from−20°C. glycerol stocks and incubated anaerobically overnight in MRS brothand washed in antibiotic containing medium. Epithelial cell monolayerswere gown for 6 weeks prior to the addition of PBMCs and bacterialcells.

The results of these stimulations can be observed in FIG. 8. Relative tocontrol cultures, two bacterial strains suppressed TNFα production. Thetwo strains Lactobacillus salivarius strain UCC118, which suppressedproduction of TNFα, is the subject of WO-A-9835014. The Bifidobacteriumlongum infantis strain UCC 35624 is the subject of a PCT Applicationfiled concurrently with the present application.

Inflammation

Inflammation is the term used to describe the local accumulation offluid, plasma proteins and white blood cells at a site that hassustained physical damage, infection or where there is an ongoing immuneresponse. Control of the inflammatory response is exerted on a number oflevels (for review see Henderson B., and Wilson M. 1998. In“Bacteria-Cytokine interactions in health and disease. Portland Press,79-130). The controlling factors include cytokines, hormones (e.g.hydrocortisone), prostaglandins, reactive intermediates andleukotrienes. Cytokines are low molecular weight biologically activeproteins that are involved in the generation and control ofimmunological and inflammatory responses, while also regulatingdevelopment, tissue repair and haematopoiesis. They provide a means ofcommunication between leukocytes themselves and also with other celltypes. Most cytokines are pleiotrophic and express multiple biologicallyoverlapping activities. Cytokine cascades and networks control theinflammatory response rather than the action of a particular cytokine ona particular cell type (Arai K I, et al., Annu Rev Biochem1990;59:783-836). Waning, of the inflammatory response results in lowerconcentrations of the appropriate activating signals and otherinflammatory mediators leading to the cessation of the inflammatoryresponse. TNFα is a pivotal proinflammatory cytokine as it initiates acascade of cytokines and biological effects resulting in theinflammatory state. Therefore, agents which inhibit TNFα are currentlybeing used for the treatment of inflammatory diseases, e.g. infliximab.

Pro-inflammatory cytokines are-thought to play a major role in thepathogenesis of many inflammatory diseases, including inflammatory boweldisease (IBD). Current therapies for treating IBD are aimed at reducingthe levels of these pro inflammatory cytokines, including IL-8 and TNFα.Such therapies may also play a significant role in the treatment ofsystemic inflammatory diseases such as rheumatoid arthritis.

In view of the anti-inflammatory properties of Lactobacillus salivariusthat we have discovered these strains may have potential application inthe treatment of a range of inflammatory diseases, particularly if usedin combination with other anti-inflammatory therapies, such asnon-steroid anti-inflammatory drugs (NSAIDs) or Infliximab.

Diarrhoeal Disease.

The barrier function of the intestinal epithelium can be diminishedduring nervous (acetylcholine) and immune (histamine) mediatedsecretion. Certain bacterial toxins may also induce Ca2+and PKCdependent secretion and thereby can disturb the epithelial barrier(Ganguly N K and Kaur T. Indian J Med Res 1996;104:28-37, Groot J. A.Vet Q 1998;20(S3):45-9). Several studies have examined the preventionand treatment of diarrhoea using probiotic bacteria. Prospective studieshave demonstrated the efficacy of lactic acid bacteria administrationfor both prophylactic and therapeutic use against diarrhoea inpre-mature infants, new borns, children (Isolauri E, et al., Dig Dis Sci1994 December;39(12):2595-600) and in the treatment of antibioticrelated diarrhoea (Siitonen S, et al., Ann Med 1990 February;22(1):57-9)and travellers diarrhoea, (Oksanen P. J, et al., Ann Med 1990February;22(1):53-6).

In view of the anti-inflammatory effect we have discovered Lactobacillussalivarius may also produce an anti-diarrhoeal effect, possiblymedicated via cAMP modulation. Cyclic AMP-dependent Cl-secretion is themajor secretory pathway in the human intestine (Brzuszczak I M, et al.,J. Gastroenterol. Hepatol. 1996;11(9):804-10). The anti-diarrhoealeffect may not be restricted just to diarrhoea resulting fromgastrointestinal inflammation, but can be applied to the generaltreatment of diarrhoea disease.

Autoimmune Disease

The immune system has a large repertoire of specificities expressed by Band T cells. Some of these specificities will be directed toself-components. Self-recognition is normally controlled by clonaldeletion and inactivation of self-reactive lymphocytes. However, thereis a constant background of autoimmunity with antibodies to manyproteins being found in serum. A breakdown in the self-nonselfrecognition system results in autoimmunity. When autoimmune disease doesoccur, the resulting immune response damages the tissue bearing theoffending antigen. Immune complex deposition, type II hypersensitivityand cell-mediated reactions are the most important mechanisms by whichimmunopathological damage occurs. Examples of autoimmune diseasesinclude, but are not limited to, systemic lupus erythematosus,rheumatoid arthritis, insulin dependent diabetes mellitus, myastheniagravis and pernicious anaemia. We have found that Lactobacillussalivarius is an immunomodulatory bacterium. Thus, consumption either asa single component or in combination with other bacteria by patientssuffering from autoimmune disease may restrict organ damage and helprestore normal body homeostasis.

Inflammation and Cancer

The production of multifunctional cytolines across a wide spectrum oftumour types suggests that significant inflammatory responses areongoing in patients with cancer. It is currently unclear what protectiveeffect this response has against the growth and development of tumourcells in vivo. However, these inflammatory responses could adverselyaffect the tumour bearing host. Complex cytokine interactions areinvolved in the regulation of cytokine production and cell proliferationwithin tumour and normal tissues (McGee D W, et al., Immunology 1995September;86(l):6-11, Wu S, et al.,. Gynecol Oncol 1994April;53(.1):59-63). It has long been recognised chat weight loss(cachexia) is the single most common cause of death in patients withcancer (Inagaki J, et al., Cancer 1974 February;33(2)-568-73) andinitial malnutrition indicates a poor prognosis (Van Eys J. Nutr Rev1982 December;40(12):353-9). For a tumour to grow and spread it mustinduce the formation of new blood vessels and degrade the extracellularmatrix. The inflammatory response may have significant roles to play inthe above mechanisms, thus contributing to the decline of the host andprogression of the tumour. Due to the anti-inflammatory properties ofLactobacillus salivarius these bacterial strains they may reduce therate of malignant cell transformation. Furthermore, intestinal bacteriacan produce, from dietary compounds, substances with genotoxic,carcinogenic and tumour-promoting activity and gut bacteria can activatepro-carcinogens to DNA reactive agents (Rowland I. R. (1995). Toxicologyof the colon: role of the intestinal microflora. In: Gibson G. R. (ed).Human colonic bacteria: role in nutrition, physiology and pathology, pp155-174. Boca Raton CRC Press). In general, species of Lactobacillushave low activities of xenobiotic metabolising enzymes compared to otherpopulations within the gut such as bacteroides, eubacteria andclostridia (Saito Y., et al., Microb. Ecol. Health Dis., 1992;5,105-110). Therefore, increasing the number of Lactobacillus bacteria inthe gut could beneficially modify the levels of these enzymes.

Prebiotics

The introduction of probiotic organisms is accomplished by the ingestionof the microorganism in a suitable carrier. It would be advantageous toprovide a medium that would promote the growth of these probioticstrains in the large bowel. The addition of one or moreoligosaccharides, polysaccharides, or other prebiotics enhances thegrowth of lactic acid bacteria in the gastrointestinal tract (Gibson, GR. Br. J. Nutr. 1998;80 (4):S209-12). Prebiotics refers to anynon-viable food component that is specifically fermented in the colon byindigenous bacteria thought to be of positive value, e.g.bifidobacteria, lactobacilli. Types of prebiotics may include thosewhich contain fructose, xylose, soya, galactose. glucose; and mannose.The combined administration of a probiotic strain with one or moreprebiotic compounds may enhance the growth of the administered probioticin vivo resulting in a more pronounced health benefit, and is termedsynbiotic.

Other Active Ingredients

It will be appreciated that the Lactobacillus salivarius may beadministered prophylactically or as a method of treatment either on itsown or with other probiotic and/or prebiotic materials as describedabove. In addition, the bacteria, may be used as part of a prophylacticor treatment regime using other active materials such as those used fortreating inflammation or other disorders, especially those, of thegastrointestinal tract. Such combinations administered in a singleformulation or as separate formulations administered at the same ordifferent times and using the same or different routes ofadministration.

The invention is not limited to the embodiments hereinbefore describedwhich may be varied in detail.

1-50. (canceled)
 51. (canceled)
 52. A method of treating or preventinginflammation or an inflammatory disease in a subject which comprisesadministering to the subject a preparation comprising a Lactobacillussalivarius strain of Lactobacillus salivarius wherein the Lactobacillussalivarius strain is of human origin.
 53. The method as claimed in claim52 wherein the Lactobacillus salivarius effects changes in animmunological marker when introduced into a system comprising cellswhich interact with the immune system and cells of the immune system.54. The method as claimed in claim 53 wherein the cells which interactwith the immune system are epithelial cells.
 55. The method as claimedin claim 53 wherein the immunological marker is a cytokine.
 56. Themethod as claimed in claim 55 wherein the cytokine is TNFα.
 57. Themethod as claimed in claim 53 wherein the cells which interact with theimmune system and the immune system cells are of matched origin.
 58. Themethod as claimed in claim 53 wherein the cells which interact with theimmune system are of an origin selected from gastrointestinal,respiratory and genitourinary.
 59. The method as claimed in claim 53wherein the cells of the immune system are of an origin selected fromgastrointestinal, respiratory and genitourinary.
 60. The method asclaimed in claim 52 wherein the Lactobacillus salivarius strain isLactobacillus salivarius subspecies salivarius.
 61. The method asclaimed in claim 52 wherein the Lactobacillus salivarius is isolatedfrom resected and washed human gastrointestinal tract.
 62. The method asclaimed in claim 52 wherein the Lactobacillus salivarius inhibits abroad range of Gram positive and Gram negative micro-organisms.
 63. Themethod as claimed in claim 52 wherein the Lactobacillus salivariussecretes a product having anti-microbial activity into a cell-freesupernatant, said activity being produced only by growing cells andbeing destroyed by proteinase K and pronase E.
 64. The method as claimedin claim 52 wherein the Lactobacillus salivarius strain is strain UCC118 [NCIMB40829].
 65. The method as claimed in claim 52 wherein theLactobacillus salivarius is a genetically modified mutant of strain UCC118 [NCIMB40829].
 66. The method as claimed in claim 52 wherein theLactobacillus salivarius is a naturally occurring variant of strain UCC118 [NCIMB40829].
 67. The method as claimed in claim 52 wherein theLactobacillus salivarius is in the form of viable cells.
 68. The methodas claimed in claim 52 wherein the Lactobacillus salivarius is in theform of non-viable cells.
 69. The method as claimed in claim 52 whereinthe undesirable inflammatory activity is undesirable gastrointestinalinflammatory activity.
 70. The method as claimed in claim 69 wherein thegastrointestinal inflammatory activity is inflammatory bowel disease.71. The method as claimed in claim 69 wherein the gastrointestinalinflammatory activity is Crohns disease.
 72. The method as claimed inclaim 69 wherein the gastrointestinal activity is ulcerative colitis.73. The method as claimed in claim 69 wherein the gastrointestinalinflammatory activity is irritable bowel syndrome.
 74. The method asclaimed in claim 69 wherein the gastrointestinal inflammatory activityis pouchitis.
 75. The method as claimed in claim 69 wherein thegastrointestinal inflammatory activity is post infection colitis. 76.The method as claimed in claim 69 wherein the inflammatory activity isdue to gastrointestinal cancer.
 77. The method as claimed in claim 52wherein the inflammatory activity is systemic inflammatory disease. 78.The method as claimed in claim 77 wherein the systemic inflammatorydisease is rheumatoid arthritis.
 79. The method as claimed in claim 52wherein the undesirable inflammatory activity is due to an autoimmunedisorder.
 80. The method as claimed in claim 52 wherein the undesirableinflammatory activity is due to cancer.
 81. The method as claimed inclaim 52 wherein the Lactobacillus salivarius is contained in aformulation.
 82. The method as claimed in claim 81 wherein theformulation includes another probiotic material.
 83. The method asclaimed in claim 81 wherein the formulation includes a prebioticmaterial.
 84. The method as claimed in claim 81 wherein the formulationincludes an ingestable carrier.
 85. The method as claimed in claim 84wherein the ingestable carrier is a pharmaceutically acceptable carrier.86. The method as claimed in claim 84 wherein the ingestable carrier isselected from one or more of a protein, a peptide, a lipid, acarbohydrate, a vitamin, a mineral, and a trace element.
 87. The methodas claimed in claim 84 wherein the ingestable carrier is a food product.88. The method as claimed in claim 81 wherein the Lactobacillussalivarius is present at more than 10⁶ cfu per gram of the formulation.89. The method as claimed in claim 81 wherein the formulation includesan ingredient selected from one or more of an adjuvant, a bacterialcomponent, a drug entity, and a biological compound.
 90. A method oftreating or preventing a diarrhoeal disease in a subject comprisingadministering to the subject a preparation comprising a Lactobacillussalivarius strain wherein the Lactobacillus salivarius strain is ofhuman origin.
 91. A method of treating or preventing inflammation or aninflammatory disease in a subject comprising administering to thesubject a preparation comprising Lactobacillus salivarius strain UCC 118[NCIMB40829].
 92. A method of treating or preventing a diarrhoealdisease in a subject comprising administering to the subject apreparation comprising Lactobacillus salivarius strain UCC 118[NCIMB40829].